Creatine transporter (SLC6A8) knockout mice display an increased capacity for in vitro creatine biosynthesis in skeletal muscle.

The present study aimed to investigate whether skeletal muscle from whole body creatine transporter (CrT; SLC6A8) knockout mice (CrT(-/y)) actually contained creatine (Cr) and if so, whether this Cr could result from an up regulation of muscle Cr biosynthesis. Gastrocnemius muscle from CrT(-/y) and wild type (CrT(+/y)) mice were analyzed for ATP, Cr, Cr phosphate (CrP), and total Cr (TCr) content. Muscle protein and gene expression of the enzymes responsible for Cr biosynthesis L-arginine:glycine amidotransferase (AGAT) and guanidinoacetate methyltransferase (GAMT) were also determined as were the rates of in vitro Cr biosynthesis. CrT(-/y) mice muscle contained measurable (22.3 ± 4.3 mmol.kg(-1) dry mass), but markedly reduced (P < 0.05) TCr levels compared with CrT(+/y) mice (125.0 ± 3.3 mmol.kg(-1) dry mass). AGAT gene and protein expression were higher (~3 fold; P < 0.05) in CrT(-/y) mice muscle, however GAMT gene and protein expression remained unchanged. The in vitro rate of Cr biosynthesis was elevated 1.5 fold (P < 0.05) in CrT(-/y) mice muscle. These data clearly demonstrate that in the absence of CrT protein, skeletal muscle has reduced, but not absent, levels of Cr. This presence of Cr may be at least partly due to an up regulation of muscle Cr biosynthesis as evidenced by an increased AGAT protein expression and in vitro Cr biosynthesis rates in CrT(-/y) mice. Of note, the up regulation of Cr biosynthesis in CrT(-/y) mice muscle was unable to fully restore Cr levels to that found in wild type muscle.

Mimtags: the use of phage display technology to produce novel protein-specific probes

In recent times the use of protein-specific probes in the field of proteomics has undergone evolutionary changes leading to the discovery of new probing techniques. Protein-specific probes serve two main purposes: epitope mapping and detection assays. One such technique is the use of phage display in the random selection of peptide mimotopes (mimtags) that can tag epitopes of proteins, replacing the use of monoclonal antibodies in detection systems. In this study, phage display technology was used to screen a random peptide library with a biologically active purified human interleukin-4 receptor (IL-4R) and interleukin-13 (IL-13) to identify mimtag candidates that interacted with these proteins. Once identified, the mimtags were commercially synthesised, biotinylated and used for in vitro immunoassays. We have used phage display to identify M13 phage clones that demonstrated specific binding to IL-4R and IL-13 cytokine. A consensus in binding sequences was observed and phage clones characterised had identical peptide sequence motifs. Only one was synthesised for use in further immunoassays, demonstrating significant binding to either IL-4R or IL-13. We have successfully shown the use of phage display to identify and characterise mimtags that specifically bind to their target epitope. Thus, this new method of probing proteins can be used in the future as a novel tool for immunoassay and detection technique, which is cheaper and more rapidly produced and therefore a better alternative to the use of monoclonal antibodies.

3T3-L1 adipocytes display phenotypic characteristics of multiple adipocyte lineages

Differentiated 3T3-L1 adipocytes are a widely used in vitro model of white adipocytes. In addition to classical white and brown adipocytes that are derived from different cell lineages, beige adipocytes have also been identified, which have characteristics of both white and brown adipocytes. Here we show that 3T3-L1 adipocytes display features of multiple adipocytes lineages. While the gene expression profile and basal bioenergetics of 3T3-L1 adipocytes was typical of white adipocytes, they responded acutely to catecholamines by increasing oxygen consumption in an UCP1-dependent manner, and by increasing the expression of genes enriched in brown but not beige adipocytes. Chronic exposure to catecholamines exacerbated this phenotype. However, a beige adipocyte differentiation procedure did not induce a beige adipocyte phenotype in 3T3-L1 fibroblasts. These multiple lineage features should be considered when interpreting data from experiments utilizing 3T3-L1 adipocytes.

Reworking direct cinema: performative display in rockumentary

early 1960s, a staple of American direct cinema. In keeping with its associations with observational direct cinema, rockumentary emphasizes showing over telling: that is, rockumentary privileges the visual capacities of documentary over patterns of exposition. While the ‘documentary display’ of rockumentary is comparable to certain features of the early ‘cinema of attractions’ it exceeds such features in its focus on performance. Typically, an emphasis within documentary theory on unmediated and unreconstructed access to the real as the basis of documentary film has not admitted a place for notions of performance before the camera. Rockumentary, with its relentless foregrounding of the performing body and the performance of musicians, revises this understanding. This essay examines rockumentary within the context of observational direct cinema as a mode centred on a documentary performative display as it operates within selected works from the 1960s to the early twenty-first century. The film theorist Brian Winston has claimed that ‘[d]irect cinema made the rock performance/tour movie into the most popular and commercially viable documentary form thus far.’ The inverse of this assessment is closer to the mark: the rockumentary turned direct cinema into a commercially viable and popular form, one which the rockumentary has at times returned to and innovatively superseded in its scopic attention to performative display.

Stress-induced peak (but not resting) metabolism correlates with mating display intensity in male guppies

Recent empirical and conceptual papers have highlighted the potential for metabolism to act as a proximate mechanism for behavior that could explain animal personality (consistency over time). Under this hypothesis, individuals with consistently high levels of behavioral activity should also have high resting metabolic rate (RMR) as it can reflect capacity to process food and generate energy. We tested for the predicted positive covariance between RMR and three behaviors that differ in energy demands in 30 male guppies, using multivariate mixed models; we repeatedly measured their activity (10 times each), courtship displays (nine times), voracity (10 times), and metabolism (four-times). Resting metabolic rate (measured overnight in respirometry trials) did not consistently differ among males, whereas initial peak metabolism measured during those same trials (R = 0.42), and all behaviors were repeatable (R = 0.33–0.51). RMR declined over time suggesting habituation to the protocol, whereas peak metabolism did not. Initial peak metabolism was negatively correlated with courtship display intensity, and voracity was positively correlated with activity, but all other among-individual correlations were not significant. We conclude that RMR does not provide a proximate explanation for consistent individual differences in behavior in male guppies, and therefore the potential for independent evolution of these physiological and behavioral traits seems possible. Finally, we identify peak metabolism as a potential measure of the stress response to confinement, which highlights the value of considering various aspects of metabolic rates recording during respirometry trials.